KR102246335B1 - Apparatus and Method for Detecting Analytes Using Surface Enhanced Raman Scattering - Google Patents

Abstract

It relates to a method and apparatus for detecting a substance using surface-enhanced Raman scattering according to the present invention, wherein the detection method according to the present invention comprises a spherical plasmonic metal core, a plasmonic metal shell having surface irregularities, and the core and shell respectively. And Raman active particles located between the core and the shell and including a self-assembled monolayer including a Raman reporter and a first receptor located on the surface of the plasmonic metal shell and capable of specifically binding to a substance to be detected; A sample that may contain a detection target substance; by contacting and irradiating excitation light, the detection target substance in the sample is detected.

Description

Apparatus and Method for Detecting Analytes Using Surface Enhanced Raman Scattering

The present invention relates to an apparatus and method for detecting a substance to be detected using surface-enhanced Raman scattering, and in detail, to provide a detection apparatus and method capable of detecting a single molecule level having excellent reliability and reproducibility.

SERS (Surface-Enhanced Raman Scattering, hereinafter SERS) spectroscopy is a spectroscopy using the phenomenon that ^{the intensity of Raman scattering rapidly increases more than 10 6} ~ 10 ⁸ times when molecules are adsorbed on the surface of metal nanostructures such as gold and silver. SERS spectroscopy, which is fused with nanotechnology that is currently developing at a very fast pace, is particularly expected to be useful as a medical sensor.

As an example, SERS spectroscopy is a measurement technology with high selectivity and high information, and is a powerful analysis method for ultra-sensitive chemical/biological/biochemical analysis.Therefore, using SERS spectroscopy, in addition to high-sensitivity DNA analysis, Research is actively being conducted to perform the initial diagnosis of various diseases including Alzheimer's disease or diabetes.

However, SERS spectroscopy has high selectivity, high information, and high sensitivity, but as the signal enhancement changes very sensitively to the gap between plasmon metals, the size or type of junction, and the distance between the hot spot and the Raman signal generator, it is measured. There is a problem of poor reliability and reproducibility.

Therefore, in order to be used in the bio field such as early diagnosis of disease, the development of Raman active particles that have a high sensitivity capable of detecting a single molecule level and generate reliable and reproducible surface-enhanced Raman scattering must be preceded. In addition, the development of a technology capable of mass-producing such Raman active particles in a short time should also be preceded.

Republic of Korea Patent Publication No. 2011-0039688

An object of the present invention is to provide a surface-enhanced Raman scattering-based detection apparatus and method capable of reproducible, reliable and quantitative detection of a detection object.

Another object of the present invention is to provide a surface-enhanced Raman scattering-based detection apparatus and method having extremely excellent sensitivity capable of detecting a single molecule level.

Another object of the present invention is to provide a surface-enhanced Raman scattering-based detection device and method suitable for detection of a biomaterial including a biochemical material because elements in contact with a detection object are biocompatible, which is used in a detection device and method.

The detection method according to the present invention includes a spherical plasmonic metal core, a plasmonic metal shell having surface irregularities, a self-assembled monolayer and the plasma which are combined with each of the core and the shell and located between the core and the shell and include a Raman reporter. Raman active particles located on the surface of the monometallic shell and including a first receptor capable of specifically binding to the detection target material; contact with a sample that may contain the detection target material, and irradiation with excitation light, Detects the substance to be detected in the sample.

In the detection method according to an embodiment of the present invention, the excitation light may be near infrared rays.

A detection method according to an embodiment of the present invention includes the steps of: a) contacting a sample with a substrate on the surface of which a second receptor specifically binds to a substance to be detected; b) contacting the Raman active particles with the substrate in contact with the sample; c) obtaining a Raman spectrum by irradiating excitation light on a substrate in contact with the Raman active particles; And d) detecting the presence or absence of the detection target substance and the concentration of the detection target substance in the sample based on the Raman spectrum.

In the detection method according to an embodiment of the present invention, step c) includes obtaining a Raman map of an Il Raman signal by performing Raman mapping with a preset area, and step d) is performed on the Raman map. The presence or absence of the detection target substance in the sample and the concentration of the detection target substance may be detected based on the sum strength obtained by summing the maximum intensity of the Il Raman signal.

In the detection method according to an embodiment of the present invention, the concentration of the detection target substance may be calculated by Equation 1 below.

(Equation 1)

MC= aI _sum + b

In Equation 1, MC is the logarithm of the molar concentration of the substance to be detected, and I _sum is the summation strength.

The detection method according to an embodiment of the present invention may further include removing unreacted samples after step a) and removing unreacted Raman active particles after step b).

In the detection method according to an embodiment of the present invention, the detection target material may be selected from one or two or more of lesion-labeled biomaterials, pathogens, proteins, nucleic acids, sugars, drugs, and biochemicals having lesion specificity.

In the detection method according to an embodiment of the present invention, the Raman map may be one in which the basal fluorescence has not been removed.

In the detection method according to an embodiment of the present invention, the plasmonic metal shell includes plasmonic metal fine particles having an average size of 0.1D to 0.6D based on the diameter (D) of the metal core, and the plasmonic metal fine particles It may have surface irregularities due to.

In the detection method according to an embodiment of the present invention, in the plasmon metal shell, the inner surface shape of the shell in contact with the self-assembled monolayer may be spherical.

In the detection method according to an embodiment of the present invention, the thickness of the self-assembled monolayer may be 0.5 to 2.0 nm.

The present invention includes a method for detecting a lesion labeling substance for diagnosing a disease.

The detection method according to the present invention is a method of detecting a lesion labeling substance for diagnosing a disease, and a biological-derived sample that may contain a lesion labeling substance and a substrate in which a second receptor specifically binding to the lesion labeling substance is located on the surface Contacting; A substrate in contact with a biological sample and a spherical plasmonic metal core, a plasmonic metal shell having surface irregularities, a self-assembled monolayer film that is bonded to each of the core and shell and is located between the core and the shell and includes a Raman reporter, and the Washing the Raman active particles located on the surface of the plasmonic metal shell and including the first receptor capable of specifically binding to the detection target material; Irradiating near-infrared excitation light on a substrate in contact with the Raman active particles and performing Raman mapping with a preset area to obtain a Raman map of an E Raman signal; And detecting the presence or absence of a lesion labeling substance in the biological sample and the concentration of the lesion labeling substance based on the summation intensity obtained by summing the maximum intensity of the Il Raman signal on the Raman map.

The present invention includes a detection device for detecting a substance to be detected using surface-enhanced Raman scattering.

The detection device according to the present invention includes a spherical plasmonic metal core, a plasmonic metal shell having surface irregularities, a self-assembled monolayer film that is coupled to each of the core and the shell and is positioned between the core and the shell and includes a Raman reporter, and the plasma. A surface-enhanced Raman scattering-active reagent comprising Raman active particles located on the surface of the monometallic shell and including a first receptor capable of specifically binding to a substance to be detected; And a substrate having a second functional group specifically binding to the detection target substance positioned on the surface.

Raman active particles used in the detection method and apparatus according to an embodiment of the present invention have a core-shell structure of a plasmon active shell having a spherical plasmon active core and surface irregularities due to fine particles. In addition, the Raman active particles are strictly defined by a self-assembled monomolecular film including a Raman reporter positioned between the core-shell, the shape of a spherical core, and a shell having a spherical inner surface by wrapping the self-assembled monomolecular film. It has a size and shape and has a hot spot uniformly present in the entire area of the particle. Along with this, Raman reporters are uniformly and densely located in the form of a self-assembled monolayer in a hot spot area where the Raman active particles are well defined and uniformly and continuously exist in the entire area of the particles. In addition, as the metal shell protrudes from the metal particles themselves and forms bumpy irregularities on the entire surface of the metal shell, one particle has uniform Raman activity, and with this, while having uniform Raman activity between particles, It can greatly improve the sensitivity of particles. Due to the characteristics and structure of the Raman active particles, the Raman active particles have uniform Raman activity on a particle basis, and have substantially the same Raman activity between particles. Accordingly, the detection method and apparatus using such Raman active particles can quantitatively detect a substance to be detected with reproducibility and reliability, and can have excellent detection capability at the level of a single molecule.

In addition, well-defined hot spots continuously exist in all regions of the Raman active particles, and Raman reporters are uniformly located at high density within the well-defined hot spots. Accordingly, the detection method and apparatus using such Raman active particles has the advantage of reproducibly detecting biochemical substances (biomaterials) having a size of several to tens of micrometers.

In addition, the detection method and apparatus according to an example of the present invention has the advantage of preventing damage to a biological sample by irradiation of excitation light for Raman analysis, since it is possible to detect a substance by irradiation with near-infrared rays of 750 nm or more.

In addition, the detection method and apparatus according to an embodiment of the present invention have a high Raman signal intensity and substantially no basal fluorescence when irradiating near-infrared rays of 750 nm or more with excitation light, so that the detection signal is subjected to post-processing. It is free from Raman signal distortion and has the advantage of having high detection reliability.

In addition, the Raman active particles used in the detection method and apparatus according to an example of the present invention are free from surfactants and thus have biocompatibility.

In addition, the Raman active particles used in the detection method and device according to an embodiment of the present invention are protected by being surrounded by an organic component including a Raman reporter by a shell, and a core-Raman reporter self-assembled monolayer-metal by a functional group. As the shell is strongly bonded, there is an advantage of having very excellent durability and physical/chemical stability.

In addition, the detection device according to an example of the present invention is excellent in mass production in a short time by a very simple method of mixing a buffer solution, a metal precursor, and a spherical core metal at room temperature without the aid of a surfactant in the Raman active particles. It has the advantage of having commerciality.

1 is a scanning electron microscope photograph of a low magnification (FIG. 1(a)) and high magnification (FIG. 1(b)) observing Raman active particles prepared according to an embodiment of the present invention.
2 is a transmission electron microscope photograph of Raman active particles prepared according to an embodiment of the present invention.
3 is a UV-Vis absorption of each of the Au core itself (cpre AuNP in FIG. 3), the Au core with a self-assembled monolayer (BDT-treated AuNP in FIG. 3), and Raman active particles prepared according to an embodiment of the present invention. It is a diagram showing the spectrum.
4 is a diagram illustrating a measurement of a Raman spectrum of a Raman active particle itself prepared according to an embodiment of the present invention.
5 is a scanning electron microscope photograph (FIG. 5(a)) and a scanning electron microscope observed after placing Raman active particles (α, β, γ) prepared according to an embodiment of the present invention on a silicon substrate. It is a diagram showing the Raman mapping (780nm laser, 5mW) of the region.
FIG. 6 is a diagram showing an overlapping Raman spectrum of each of Raman active particles α, β, and γ mapped to Raman in FIG. 5.
7 is a diagram showing a Raman spectrum (FIG. 7(a)) and a Raman map (FIG. 7(b)) obtained according to an embodiment of the present invention using tau protein as a detection target material.
8 is a schematic diagram showing a structure in which tau protein is bound to a second receptor of a tau substrate and a tau probe is bound to the tau protein bound to the substrate according to an embodiment of the present invention.
9 is a diagram illustrating Raman mapping results according to the concentration of tau protein in a sample according to an embodiment of the present invention.
FIG. 10 is a reference graph calculated from a standard sample (Tau in PBS in FIG. 10) with different tau protein concentrations and cerebrospinal fluid (Tau in aCSF in FIG. 10) or plasma ( Tau in plasma of FIG. 10) is a diagram showing a result of detecting the sample.

Hereinafter, the detection method and apparatus of the present invention will be described in detail with reference to the accompanying drawings. The drawings introduced below are provided as examples in order to sufficiently convey the spirit of the present invention to those skilled in the art. Accordingly, the present invention is not limited to the drawings presented below and may be embodied in other forms, and the drawings presented below may be exaggerated to clarify the spirit of the present invention. At this time, unless there are other definitions in the technical terms and scientific terms used, they have the meanings commonly understood by those of ordinary skill in the technical field to which the present invention belongs, and the gist of the present invention in the following description and accompanying drawings Descriptions of known functions and configurations that may be unnecessarily obscure will be omitted. In addition, the singular form used in the specification and the appended claims may be intended to include the plural form unless otherwise indicated in the context. Units used in this specification and the appended claims are based on weight, and as an example, the unit of% or ratio means weight% or weight ratio.

In the detection method according to the present invention, the Raman active particles are brought into contact with a sample that may contain the detection target material, and excitation light is irradiated to detect the detection target material in the sample.

In one embodiment, the Raman active particles are Raman active particles for surface-enhanced Raman scattering (SERS), including a spherical plasmonic metal core; A plasmonic metal shell having surface irregularities; A self-assembled monolayer film coupled to each of the core and the shell, positioned between the core and the shell, and including a Raman reporter; And a first receptor positioned on the surface of the plasmonic metal shell and capable of specifically binding to a substance to be detected.

The self-assembled monolayer disposed between the core and the shell has a strictly controlled thickness due to the characteristics of self-assembly. Accordingly, a strictly defined nanogap having a size corresponding to the thickness of the self-assembled monolayer may be formed between the core and the shell. In addition, nanogaps (hot spots) having a uniform size may be formed in the entire area of the Raman active particles due to the structure of the core-self-assembled monolayer-shell.

In addition, as the shape of the plasmonic metal core is spherical, the self-assembled monolayer has a spherical shape, and the plasmonic metal shell may also have a spherical shape of the inner surface of the metal shell in contact with the self-assembled monolayer. Accordingly, the nanogap (hot spot) may be located in the entire area of the Raman active particle, and the nanogap (hot spot) may be located at the same well-defined position in all directions based on the radial direction.

In addition, in the Raman active particles, as the self-assembled monomolecular film positioned between the core and the shell contains a Raman reporter, the Raman reporter is well defined in the Raman active particles and is located at the same position in the radial direction, and the entire area of the Raman active particles The Raman reporter is located at high density and uniformly, and the Raman reporter is located at the hot spot.

Such Raman active particles may exhibit uniform SERS activity on a particle basis, and there is little variation in Raman activity between particles, so that uniform SERS activity between particles may be exhibited, and a greater Raman signal may be enhanced.

As a specific example of uniform SERS activity, on the Raman spectrum of the Raman active particles, the standard deviation (a.u.) of the Raman signal intensity (maximum intensity of the Il Raman signal, a.u.)) may be 8.0 or less. At this time, the Raman spectrum of the Raman active particles may be performed by irradiating near-infrared light, and may be performed using a known type of Raman spectroscopy. For example, Raman spectroscopy may be obtained under conditions of a 780nm laser, a laser power of 5mW, an N.A.0.75 objective lens, and a laser exposure time of 1 second. The standard deviation may be calculated from Raman spectra of 50 or more Raman active particles, but is not limited thereto.

In one embodiment, the plasmon metal core and the plasmon metal shell may be metals in which surface plasmons are generated by interaction with light, respectively. For example, the plasmon metal core and the plasmon metal shell may be each of gold, silver, platinum, palladium, nickel, aluminum, copper, or a mixture thereof or an alloy thereof. However, in consideration of biocompatibility, the plasmon metal core and the plasmon metal shell may be gold or silver, respectively.

In one embodiment, the plasmonic metal shell includes plasmonic metal fine particles having an average size of 0.1D to 0.6D based on the diameter (D) of the metal core, and may have surface irregularities due to the plasmonic metal fine particles. . Specifically, the metal shell in a state combined with the self-assembled monolayer may be made of metal particles having an average size of 0.1D to 0.6D based on the diameter (D) of the metal core, and the metal shell is It may have irregular irregularities.

The concave-convex structure of the metal fine particles of the plasmonic metal shell is more advantageous for signal enhancement because hot spots can be formed on the surface of the shell itself as well as hot spots due to the nanogap between the metal core and the metal shell. In addition, as the metal shell protrudes from the metal particles themselves and forms bumpy irregularities on the entire surface of the metal shell, the sensitivity of the Raman active particles can be enhanced by the metal shell, as well as in one particle. It may not inhibit the uniformity of Raman activity and the uniformity of Raman activity between particles.

The average diameter of the plasmon metal core may be 20 to 100 nm, specifically 20 to 80 nm, and more specifically 30 to 70 nm, but is not limited thereto.

In one embodiment, the self-assembled monolayer may be a self-assembled monolayer of a Raman reporter. The Raman reporter may refer to an organic compound (organic molecule) including a Raman active molecule, and may refer to an organic compound (organic molecule) including a Raman active molecule having a binding force with a metal of a metal core. Raman reporters are well known, and any ones widely used in this technical field may be used.

As the Raman reporter (molecule) has a bonding force with the metal of the metal core, a self-assembled monolayer of the Raman reporter may be formed on the metal core to which the pure metal surface is exposed.

The Raman active molecule may include a surface enhanced Raman active molecule, a surface enhanced resonance Raman active molecule, a hyper Raman active molecule, or a coherent van Stokes Raman active molecule. The Raman active molecule may have a Raman signal and a fluorescent signal at the same time or may have a Raman signal.

In a specific embodiment, the Raman active molecule is cyanines, fluorescein, rhodamine, 7-nitrobenzene-2-oxa-1,3-diazole (7-nitrobenz-2-oxa- 1,3-diazole: NBD), phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, paraaminobenzoic acid, Erythrosine, biotin, dioxygenin, phthalocyanine, azomethine, xanthine, N,N-diethyl-4-(5′-azobenzotriazolyl)-phenylamine, aminoacridine, and It may be selected from the group consisting of a combination thereof. Examples of cyanine may include Cy3, Cy3.5, or Cy5. Examples of fluorescein include carboxyfluorescein (FAM), 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein (6-carboxy-2', 4,4′,5′,7,7′-hexachlorofluorescein: HEX), 6-carboxy-2′,4,7,7′-tetrachlorofluorescein (6-carboxy-2′,4,7,7 ′-Tetrachlorofluorescein: TET), 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxy fluorescein, 6-carboxy-4′,5′-dichloro-2′, 7′-dime Oxyfluorescein (6-carboxy-4′,5′-dichloro-2′, 7′-dimethoxyfluorescein: Joe) 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5 -May contain carboxyfluorescein, or succinylfluorescein. Examples of rhodamine include tetramethylrhodamine (Tamra), 5-carboxyrhodamine, 6-carboxyrhodamine, 6G (Rhodamine 6G: R6G), tetramethyl rhodamine isothiol (TRIT), Sulforhodamine 101 acid chloride (Texas Red dye), carboxy-X-rhodamine (Rox), or rhodamine B (rhodamine B) may be included.

In another embodiment, the Raman active molecule may be a Raman active molecule in the form of a benzene ring, and the Raman active molecule in the form of a benzene ring is 4-Aminothiophenol (4-ATP), 4-Mercaptobenzoic acid (4-MBA), Phenyl isothiocyanate ( PITC), Benzenethiol(BT), 1,4-Benzenedithiol(BDT), Biphenyl-4,4'-dithiol(BPDT), p- Terphenyl-4,4''-dithiol(TPDT), 4-Bromobenzenethiol(4- BBT), 4-Chlorobenzenethiol (4-CBT), 3,3'-Diethylthiatricarbocyanine iodoide (DTTC), and the like.

However, as a nano-gap (hot-spot) between the metal core and the metal shell is formed by the Raman reporter coupled to the metal core, the length (size) of the Raman reporter is 3 nm or less in terms of hot spot formation in which stronger signal enhancement is made, Specifically, it is preferably 0.5 to 2 nm. In this case, it goes without saying that the length (size) of the Raman reporter corresponds to the thickness of the seat-assembled monolayer.

In addition, the Raman reporter includes a Raman active molecule, but preferably has a first functional group that spontaneously binds to the metal core. Even better, it is preferable to have a first functional group that spontaneously bonds with the metal of the metal core and a second functional group that spontaneously bonds with the metal of the metal shell. In this case, the self-assembled monolayer may be chemically bonded to each of the metal core and the metal shell, thereby greatly improving the bonding strength between the metal shell and the metal core to which the Raman reporter is fixed.

The functional group (first functional group or second functional group) may be any functional group known to spontaneously bond with the metal in consideration of the metals of the core and the shell. As a practical example, when the first metal and the second metal are independently gold or silver, the functional group (the first functional group or the second functional group) is a thiol group (-SH), a carboxyl group (-COOH), or an amine group (-NH ₂ ), etc., but the present invention is not limited by the specific types of functional groups.

By spontaneously binding (fixing) Raman active molecules with the metal of the metal core to the metal core by the first functional group, a self-assembled monolayer of the Raman reporter can be formed on the metal core. The film can be formed homogeneously over the entire surface of the metal core.

Raman active particles may include a receptor that is immobilized on a plasmonic metal shell and capable of specifically binding to a substance to be detected. The receptor may be any substance known to specifically bind to a substance to be detected, such as an enzyme-substrate, an antigen-antibody, a protein-protein, or a complementary binding between DNA. At this time, the receptor may include a functional group (for example, a thiol group, a carboxyl group, or an amine group) that spontaneously binds to the metal of the metal shell, and may be spontaneously and chemically bonded to the metal shell by the functional group. . If necessary, the metal shell may be in a surface-modified state to react with and bind to a functional group of the receptor, or to bind to a blocking molecule described below. For example, the surface of the metal shell may be a surface modified with a carboxyl group, a thiol group, an amine group, or a hydroxy group.

In one embodiment, the Raman active particles may further include blocking molecules that cover the surface area of the shell to which the receptor is not attached (bound). The blocking molecule prevents unwanted interaction between the shell surface itself and the substance to be detected, not the receptor, and may play a role in making the orientation of the receptor located on the surface of the shell more uniform. The blocking molecule is sufficient as long as it is a material commonly used to prevent non-specific binding on the metal surface in the field of biosensors, such as bovine serum albumin (SBA).

The substance to be detected may be a biological material (including a virus) or a non-living material. Specifically, the substance to be detected may include a lesion-labeled biomaterial, pathogen, protein, nucleic acid, sugar, drug, biochemical substance, etc. having lesion specificity. More specifically, the substances to be detected are amino acids, peptides, polypeptides, proteins, glycoproteins, lipoproteins, nucleosides, nucleotides, oligonucleotides, nucleic acids, sugars, carbohydrates, oligosaccharides, polysaccharides, fatty acids, lipids, Hormones, metabolites, cytokines, chemokines, receptors, neurotransmitters, antigens, allergens, antibodies, substrates, metabolites, cofactors, inhibitors, drugs, pharmaceuticals, nutrients, prions, toxins, poisons, explosives, pesticides, chemistry It may be an inorganic agent, a biohazard agent, a radioisotope, a vitamin, a heterocyclic aromatic compound, a carcinogen, a mutagenic agent, an anesthetic agent, amphetamine, barbiturate, hallucinogen, waste or contaminant. In addition, when the analyte is a nucleic acid, the nucleic acid may include genes, viral RNA and DNA, bacterial DNA, fungal DNA, mammalian DNA, cDNA, mRNA, RNA and DNA fragments, oligonucleotides, synthetic oligonucleotides, modified oligonucleotides, Single-stranded and double-stranded nucleic acids, natural and synthetic nucleic acids, and the like.

The substance to be detected may be located in-vivo, and may be detected in the living body. That is, the aforementioned Raman active particles may be for in-vivo use or for in-vivo injection.

Unlike this, the substance to be detected may be located outside the body and may be detected outside the body. That is, the above-described Raman activator may be for in-vitro use. In this case, the substance to be detected may be in the form of a sample collected from a living body, such as blood, urine, mucosal detachment, saliva, body fluid, tissue, biopsy material, or a combination thereof, but is not limited thereto.

In one embodiment, the Raman active particles are for near-infrared excitation light having a wavelength of 750 nm or more, specifically for near-infrared excitation light belonging to the 750 nm to 1500 nm wavelength band, more specifically for the 750 to 1000 nm wavelength band, 770 nm to 1500 nm wavelength band, or 780 nm to It may be for near-infrared excitation light belonging to the 1000 nm wavelength band. That is, the Raman active particles may be detected and analyzed for a substance to be detected by irradiation with light in the near-infrared band.

As is known, when visible light is irradiated to biomaterials including biochemicals, fluorescence may occur. The intensity of fluorescence is very strong compared to Raman scattering, and since fluorescence occurs in a region similar to Raman scattering, there is a problem that it is difficult to obtain a pure Raman spectrum by covering the fluorescence peak. Accordingly, SERS analysis by irradiation in the near-infrared band other than visible light can obtain Raman spectra without the influence of fluorescence, which is very advantageous in the bio field.

Substantially, upon detection of a substance to be detected using Raman active particles according to an exemplary embodiment, basal fluorescence may not appear substantially in the Raman spectrum of the substance to be detected obtained by near-infrared irradiation.

Accordingly, the Raman spectrum obtained by near-infrared irradiation may not be removed from fluorescence. That is, the presence or absence of the detection target material and the Raman spectrum used for quantitative analysis may be a spectrum detected by a Raman detector and a spectrum detected by a Raman detector and not modified by post-processing.

However, the Raman active particles of the present invention are limited to near infrared rays and should not be interpreted, and the excitation light irradiated by the detection method of the present invention is limited to near infrared rays and should not be interpreted. _{For example, based on the center wavelength (λ max} ) of the maximum absorption peak in the ultraviolet-visible absorption spectrum of the Raman active particles, light belonging to the center wavelength (λ _max ) ± 150 nm wavelength band may be irradiated as excitation light. In this case, a Raman spectrum in which a larger Raman signal is augmented can be obtained. In one embodiment, light belonging to a wavelength band of 500 to 750 nm, a wavelength of 500 to 750 nm, or a wavelength of 550 to 700 nm, or a wavelength of 600 to 680 nm may be irradiated as excitation light.

As described above, in the detection method according to the present invention, the detection target material may be detected by contacting the Raman active particles with a sample that may contain the detection target material and irradiating excitation light. Specifically, the detection method according to the present invention comprises the steps of: obtaining a Raman spectrum by contacting a Raman active particle with a sample that may contain a substance to be detected and irradiating excitation light; And detecting (quantitative analysis) a substance to be detected in the sample based on the obtained Raman spectrum.

As a specific example, the excitation light may be light belonging to a visible light to a near-infrared band. As described above, when the excitation light is near-infrared, the excitation light may be a near-infrared ray belonging to a 750 nm to 1500 nm wavelength band, a 750 to 1000 nm wavelength band, a 770 nm to 1500 nm wavelength band, or a 780 nm to 1000 nm wavelength band. In contrast, when the excitation light belongs to the visible light band, the center wavelength (λ _max ) ± 150 nm wavelength band, center wavelength _{based on the center wavelength (λ max} ) of the maximum absorption peak in the ultraviolet-visible light absorption spectrum of the Raman active particles. It may be visible light belonging to the (λ _max ) ± 100 nm wavelength band or the center wavelength (λ _{max) ± 50 nm wavelength band.}

In a specific embodiment, the detection method includes: a first step of contacting a sample with an active surface of a substrate that provides an active surface on which a second receptor capable of specifically binding to a substance to be detected is formed; A second step of bringing Raman active particles into contact with the active surface in contact with the sample; A third step of obtaining a Raman spectrum by irradiating excitation light on the active surface in contact with the Raman active particles; And a fourth step of detecting the presence or absence of the detection target substance and the concentration of the detection target substance in the sample based on the Raman spectrum. The second receptor may specifically bind to the substance to be detected, and when the substance to be detected is present in the sample, the substance to be detected may be immobilized on the active surface by the second receptor. In this case, it goes without saying that the first receptor and the second receptor can specifically bind to different sites of the substance to be detected.

The active surface of the substrate may be a metal surface such as gold, silver, platinum, palladium, nickel, aluminum, or copper, but is not limited thereto. However, when the sample is a bio-derived sample or a bio sample, it may be gold, silver, platinum, etc. having biocompatibility. The second receptor may include a functional group (for example, a thiol group, a carboxyl group, or an amine group) that spontaneously bonds to the active surface, and may be chemically bonded to the active surface by the functional group. If necessary, the active surface may react with the functional group of the second receptor to bind or may be in a surface-modified state so that it may bind to a blocking molecule to be described later. For example, the active surface may be a surface modified with a carboxyl group, a thiol group, an amine group, or a hydroxy group.

The contact in the first step may be performed by applying a liquid sample (a liquid sample or a liquefied sample) to the active surface or by immersing the active surface in the liquid sample. The applied liquid sample may be removed after a sufficient time has passed for the detection target substance that may exist in the sample to stably bind to the second receptor.

The second step of contacting may be performed by applying the Raman active particle dispersion to the active surface in contact with the sample, or by immersing the active surface in the Raman active particle dispersion. After a sufficient time has passed for the Raman active particles to stably bind to the substance to be detected immobilized on the active surface, the unreacted Raman active particles may be removed.

When the sample is applied and the Raman active particle dispersion is applied, a predetermined amount may be applied, and the applied Raman active particle dispersion may contain Raman active particles at a constant concentration. In addition, when the dispersion medium or sample of the Raman active particle dispersion liquid is liquefied, the solvent or dispersion medium used for liquefaction may be a material that does not chemically react with the Raman active particles or sample and does not affect Raman measurement. When analyzing a bio-derived sample or biomaterial, as an example of a dispersion medium of Raman active particles or a material used for liquefaction of the sample, a buffer solution such as a HEPES solution or deionized water may be mentioned, but the present invention is limited thereto. It is not.

Removal of unreacted liquid samples (residual liquid samples) applied to the active surface or removal of unreacted Raman active particles (residual Raman active particles) can be performed using a cleaning solution that does not adversely affect the analyte and Raman active particles. It can be done by washing. When analyzing a bio-derived sample or a bio material, an example of a washing solution used for washing may include deionized water, but the present invention is not limited thereto.

By the second step, the Raman active particles may specifically bind to the detection target material immobilized on the active surface, thereby forming a bonding structure of the active surface-second receptor-detection target material-Raman active particles.

In the third step, the excitation light (irradiation light) is light belonging to the visible to near-infrared band, advantageously, near infrared having a wavelength of 750 nm or more, specifically, near infrared having a wavelength of 750 nm to 1500 nm, more specifically, a wavelength of 750 to 1000 nm, 770 nm It may be a near-infrared ray having a wavelength of to 1500 nm, or a wavelength of 780 to 1000 nm.

In the fourth step, based on the obtained Raman spectrum, the presence or absence of the detection target substance in the sample and the concentration of the detection target substance may be calculated. In this case, the Raman spectrum obtained by the near-infrared irradiation in the third step may be a spectrum in which the basal fluorescence has not been removed. That is, the presence or absence of the detection target material and the Raman spectrum used for quantitative analysis may be a spectrum detected by the Raman detector and not processed by post-processing.

In a specific example, the third step may be a step of obtaining a Raman map of the E Raman signal by performing Raman mapping with a preset area, and the fourth step is to determine the maximum intensity for the E Raman signal on the Raman map. It may be a step of detecting the presence or absence of the detection target substance and the concentration of the detection target substance in the sample based on the summed strength.

The one Raman signal (one Raman wavelength) constituting the Raman map may be a signal having the highest peak intensity in the Raman spectrum of the detection target material, or a signal having the narrowest half width of the peak, but is not limited thereto.

The Raman mapping may be a Raman mapping for an area of a preset size (area of an active surface), and the preset size may be 1 to 100 μm x 1 to 100 μm, but is not limited thereto. In addition, during Raman mapping, the fapping interval may be at the level of 0.1 μm to 10 μm for each of the axes perpendicular to each other, and the output of excitation light (excitation laser light) may be at the level of 1 mW to 90 mW, for example, 1 mW to 10 mW, and excitation The light irradiation time may be 0.5 to 10 seconds, and the number of scans may be 1 to 5, but is not limited thereto.

In the fourth step, the concentration of the detection target substance may be quantified by summing the intensity of the Raman signal present on the Raman map. In this case, the summed intensity may be a maximum intensity value of a corresponding Raman signal (a maximum intensity value of a Raman peak).

That is, a specific Raman peak that exists only on the Raman map by Raman active particles having isotropic SERS activity and highly uniform SERS activity between Raman active particles, substantially no basal fluorescence, and reliability showing excellent Raman signal intensity. Quantitative analysis of the substance to be detected is possible only by summing the strengths of the fields (first peaks), and the limit of detection is 20 aM, which is extremely high to detect even a single substance to be detected. Can have sensitivity.

In practice, in a semi-log graph in which the log value of the molar concentration of the substance to be detected is the x-axis and the sum (au) of the Raman signal intensities of the specific Raman peaks (first peaks) is the y-axis, the detection target The logarithmic value (MC) of the molar concentration of the substance and the sum of the Raman signal intensity (I _sum ) may have a linear relationship. That is, on the semi-log graph, MC = aI _sum + b (a and b are each constant). In this case, this linearity can be maintained in a broad molar concentration range of ^{10 -2} fM to 10 ^{6 fM.}

Therefore, the detection method is to perform Raman mapping using standard samples containing the detection target material at a predetermined molar concentration before the first step, and obtain the sum of the Raman signal intensities at a specific Raman signal at the corresponding concentration on the Raman map. By performing the step, a step of obtaining a reference graph, which is a relational expression between the sum of the molar concentration of the substance to be detected and the Raman signal intensity, on the semi-log graph. By substituting the intensity (summed intensity) calculated in the fourth step on the reference graph, the amount of the detection target substance in the sample can be quantitatively analyzed.

The present invention includes a method for detecting a lesion labeling substance for diagnosing a disease. The method of detecting a lesion labeling substance may correspond to a method of diagnosing a disease using a lesion labeling substance. In one embodiment, the disease (including a disorder) is an infectious disease, a proliferative disease, a neurodegenerative disease, a cancer, a psychological disease, a metabolic disease, an autoimmune disease, a sexually transmitted disease, a gastrointestinal disease, a lung disease, a cardiovascular disease. , Stress-related disorder, fatigue-related disorder, fungal disease, pathogenic disease or obesity-related disorder, but is not limited thereto.

The method for detecting a lesion labeling substance according to the present invention includes the steps of contacting a substrate on which a second receptor specifically binding to the lesion labeling substance is located on a surface and a biologically-derived sample that may contain the lesion labeling substance; A substrate in contact with a biological sample and a spherical plasmonic metal core, a plasmonic metal shell having surface irregularities, a self-assembled monolayer film that is bonded to each of the core and shell and is located between the core and the shell and includes a Raman reporter, and the Washing the Raman active particles located on the surface of the plasmonic metal shell and including the first receptor capable of specifically binding to the detection target material; Irradiating near-infrared excitation light on a substrate in contact with the Raman active particles and performing Raman mapping with a preset area to obtain a Raman map of an E Raman signal; And detecting the presence or absence of a lesion labeling substance in the biological sample and the concentration of the lesion labeling substance based on the summation intensity obtained by summing the maximum intensity of the Il Raman signal on the Raman map.

The biologically derived sample may be, but not limited to, blood, blood products, serum, plasma, other blood fractions, tissues, tissue extracts, urine, cerebrospinal fluid, saliva, feces, skin, hair, cheek tissue, organ tissue, breath, pleural fluid, sweat or It may include sputum, but is not limited thereto. The biologically-derived sample may be liquid by itself, or may be liquefied if it is not. The liquefied biologically-derived sample is an enzyme or protease, a protein, a compound or preservative, a chemical stabilizer, a diluent, a buffer, an additive, for processing and increasing the half-life of the sample collected from the living body, which is commonly used for the treatment of the sample. Detergents, lipids, sugars, carbohydrates or any combination thereof may further be included. For example, enzymes such as mucin and protease may dissolve the collected sample.

The lesion labeling substance may be any atom, chemical substance, molecule, compound, or aggregate associated with a disease, which is used to determine whether a subject from a living body has a disease or not, or used to determine the degree of disease progression. have. For example, lesion markers include amino acids, peptides, polypeptides, proteins, glycoproteins, lipoproteins, nucleosides, nucleotides, oligonucleotides, nucleic acids, sugars, carbohydrates, fatty acids, lipids, hormones, metabolites, neurotransmitters, It may be an antibody or the like.

Raman spectra can be obtained using a conventional Raman detection device. As a non-limiting example, the excitation light may pass through the confocal optics and the microscope lens to be focused onto the active surface. When a substance to be detected is present on the active surface, Raman light emitted from the substance to be detected is collected by a microscope lens and a confocal optic and may be combined with a monochromatic device for spectral separation. The Raman signal can be detected by a Raman detector interfaced with a computer that counts and digitizes the signal.

The present invention includes a detection device comprising the above-described Raman active particles.

The detection device according to the present invention is a detection device for a detection target substance using surface-enhanced Raman scattering, and is combined with a spherical plasmonic metal core, a plasmonic metal shell having surface irregularities, the core and the shell, and between the core and the shell. Surface-enhanced Raman scattering comprising Raman active particles including a self-assembled monomolecular film located at and including a Raman reporter and a first receptor located on the surface of the plasmonic metal shell and capable of specifically binding to a substance to be detected- Active reagents; And a substrate in which a second functional group specifically binding to the substance to be detected is located on the surface (active surface).

In one embodiment, the surface of the substrate (active surface) or the surface of the metal shell may further include a blocking molecule covering a surface area to which a receptor (first or second receptor) is not attached (bound). The blocking molecule prevents unwanted interaction between the surface of a substrate or a metal shell other than a receptor and a substance to be detected, and may play a role of making the orientation of the receptor located on the surface of the substrate or metal shell more uniform. The blocking molecule is sufficient as long as it is a material commonly used to prevent non-specific binding on the metal surface in the field of biosensors, such as bovine serum albumin (SBA).

The present invention includes the above-described method for producing Raman active particles. Hereinafter, the manufacturing method according to the present invention will be described in detail. At this time, the metal core, the Raman reporter, the self-assembled monolayer, the metal shell, the detection target material, and the receptor are similar to or the same as described above for the Raman active particles. Accordingly, the method for producing Raman active particles according to the present invention includes all the above-described Raman active particles.

The method for preparing Raman active particles according to the present invention is a method for preparing Raman active particles for surface-enhanced Raman scattering (SERS). The manufacturing method according to the present invention comprises the steps of: a) forming a self-assembled monolayer including a Raman reporter on a spherical plasmonic metal core; And b) forming a plasmonic metal shell having surface irregularities while surrounding the metal core on which the self-assembled monolayer is formed by using a reaction solution in which the buffer solution, the metal core on which the self-assembled monolayer is formed, and the precursor of the plasmonic metal are mixed; Includes.

The method for producing Raman active particles according to the present invention has reproducibility and reliability through a simple process at low cost, has sensitivity capable of detecting single molecules, and can mass-produce biocompatible Raman active particles without a separate post-treatment.

As is known, organic surfactants capable of inhibiting growth or inducing growth in a specific direction and/or stabilizing nanoparticles while providing appropriate reducibility while providing adequate reducibility are commonly used for metal nanoparticle formation and designed shaping, Along with this, an organic acid is used, or an organic acid capable of replacing a surfactant is used. However, in the metal nanoparticles synthesized by this method, organic surfactants (or organic surfactants and organic substances derived from organic acids), which are harmful to the living body and may affect biochemical substances, are combined. Therefore, in order to be used in the bio field, a post-treatment process such as capping the particles with a capping material having biocompatibility or replacing a harmful surface functional group such as an organic surfactant with another functional group having biocompatibility is required.

However, capping using a capping material can greatly reduce the strength of biosensing or bioimaging based on SERS spectroscopy, and when an organic surfactant is to be substituted with a biocompatible functional group, an organic interface that is bonded with a metal material with a very strong binding force. Since it is difficult to completely replace the activity, there is a problem that toxicity remains.

The method for producing Raman active particles according to the present invention described above includes a buffer solution and a metal precursor having biocompatibility after forming a self-assembled monolayer including a Raman reporter on a metal core having a bare metal surface. As the metal shell is formed using the liquid, the prepared Raman active particles are free from organic surfactants that are harmful to the living body, and thus have the advantage of having biocompatibility in a state immediately after preparation.

Accordingly, in the method for producing Raman active particles according to an embodiment of the present invention, the reaction solution may not contain a surfactant (organic surfactant), and further, the reaction solution does not contain both a surfactant and an organic acid. May not.

In addition, according to the method for producing Raman active particles according to the present invention, Raman active particles are prepared using a simple process of attaching a Raman reporter to a metal core and forming a metal shell using a solution containing a buffer solution and a metal precursor, Raman active particles can be mass-produced in a short time at low cost, and thus have excellent commerciality.

In addition, in the method for producing Raman active particles according to the present invention, since the organic material including the Raman reporter is not exposed on the surface of the Raman active particle and is surrounded by a metal shell, the organic material including the Raman reporter is stable from the external environment. There is an advantage that can be protected with.

In one embodiment, the step (b)) of forming a self-assembled monolayer including a Raman reporter on a metal core includes: preparing a mixed solution containing a metal core and a Raman reporter and stirring ultrasonically; includes; can do.

Specifically, step a) comprises: a1) preparing a mixed solution by mixing the metal core and the Raman reporter to have a molar concentration of 0.01 to 1 nM and 10 to 1000 μM; a2) ultrasonically agitating and reacting at room temperature for 10 to 30 minutes; And a3) separating and recovering the metal core to which the Raman reporter is fixed. In this case, the mixed solution may be an aqueous mixed solution, but is not limited thereto.

After step a) is performed, b) a metal shell enclosing the metal core to which the Raman reporter is fixed from the reaction solution in which the buffer solution, the metal core on which the Raman reporter is fixed (the metal core on which the self-assembled monolayer is formed), and the metal precursor are mixed. The forming step can be performed. The metal core to which the Raman reporter is fixed may be a metal core on which the self-assembled monolayer of the Raman reporter is formed.

In step b), the molar ratio of the buffer solution to the metal precursor (the molar ratio obtained by dividing the number of moles of the buffer by the number of moles of the metal precursor) may be 10 to 100, preferably 20 to 80. By controlling the molar ratio to 10 to 100, preferably 20 to 80, a thin metal shell can be formed that completely surrounds the Raman reporter fixed to the metal core, and a metal shell having surface irregularities can be formed by metal particles. have.

The buffer solution is HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES(4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid), PBS(Phosphated buffered saline), Tris(2-Amino-2hydroxymethyl). propne-1,3-idol), PB(Phosphate buffer), MOPS(3-(N-morpholino)propanesulfonic acid), TAPS(3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl) ]amino]propane-1-sulfonic acid) and PIPES (piperazine-N,N'-bis (2-ethanesulfonic acid)). The buffering agent of such a buffer solution may act as a weak reducing agent to reduce the metal, and may exclude a surfactant for stabilization of Raman active particles prepared by the buffering agent of the buffer solution.

The metal of the metal precursor includes gold, silver, platinum, palladium, nickel, aluminum, copper, or a mixture thereof or an alloy thereof. However, in consideration of biostability, the metal of the metal precursor is preferably gold or silver independently of the metal of the metal core. The metal precursor according to an advantageous example may be a gold precursor such as HAuCl ₄ , HAuBr ₄ , NaAuCl ₄ , AuCl _{3 ·} 3H ₂ O, NaAuCl _{4 ·} 2H ₂ O, or a mixture thereof, or, such as _{AgNO 3} Silver may be a precursor, but is not limited thereto.

In one embodiment, in step b), a buffer solution, a metal precursor solution, and a metal core dispersion having a Raman reporter are mixed to prepare a reaction solution, and a temperature of 15 to 40° C., specifically, a temperature of 15 to 35° C. , More specifically, the reaction may be performed at a temperature of 15 to 25°C, and even more specifically at room temperature (21 to 25°C). The reaction solution may be reacted for 10 to 50 minutes, specifically 20 to 40 minutes, to prepare a metal shell, but the present invention is not limited by the reaction time of the reaction solution. At this time, stirring may be performed during the reaction, and the reaction may be terminated by adding an excess of water to the reaction solution.

The molar concentration of the buffer in the buffer solution may be 10 to 100 mM, the molar concentration of the metal precursor in the metal precursor solution may be 1 to 10 mM, and the molar concentration of the metal core in the metal core dispersion in which the Raman reporter is fixed may be 0.01 to 0.5. It may be nM, but is not limited thereto.

The buffer solution and the metal precursor solution may be mixed to satisfy the molar ratio between the buffer and the metal precursor described above, and the metal core dispersion may be mixed so that the molar ratio of the metal precursor: the metal core is 1: 1x10 ^-7 to 1x10 ^-5 . . In this case, the metal precursor solution and the metal core dispersion may be first mixed and then the buffer solution may be mixed so that the metal shell may be uniformly formed on the metal core(s).

In detail, step b) includes: b1) preparing a precursor-metal core mixture by mixing a metal precursor solution and a metal core dispersion; b2) preparing a reaction solution by mixing a buffer solution with a precursor-metal core mixture solution, and reacting the reaction solution at a temperature of 15 to 40° C., advantageously at room temperature, to prepare Raman active particles; And b3) separating and recovering the prepared Raman active particles, and adding the recovered Raman active particles to a buffer solution (separate buffer solution), and storing them at a temperature of 1 to 10°C, specifically 1 to 5°C; It may include.

By step b), Raman active particles including a metal core, a self-assembled monolayer of a Raman reporter surrounding the metal core, and a metal shell surrounding the self-assembled monomolecular film may be prepared, and have an average size of 150 nm or less, specifically, an average of 100 nm or less. Raman active particles having a size of, substantially 40 to 100 nm, and more substantially 60 to 100 nm, may be prepared.

In one embodiment, the method for producing Raman active particles may further include: after step b), c) fixing a receptor that binds (specifically binds) to a substance to be detected on a metal shell. Step c) may be performed by mixing the receptors in the prepared Raman active particle dispersion, and of course, the fixation may be performed according to a protocol known for each receptor.

In addition, before step a), a step of washing the metal core using an organic solvent or the like may be further performed so that the spherical metal core has a bare metal surface, but it is sufficient to perform such cleaning as necessary. .

The present invention includes Raman active particles prepared by the above-described manufacturing method.

1 is a scanning electron microscope photograph of a low magnification (FIG. 1(a)) and high magnification (FIG. 1(b)) observing Raman active particles prepared according to an embodiment of the present invention.

Specifically, Raman active particles were mixed with 1 mL of a 1mM BSPP (Bis (p-sulfonatophenyl) phenylphosphine dihydrate dipotassium salt) solution by using spherical Au nanoparticles (diameter = 50 nm) as a metal core, and subjected to ultrasonic treatment for 10 minutes to obtain 0.1 nM molar concentration. A dispersion of Au core was prepared. After mixing 1 mL of Au core dispersion and 50 μL of a 10 mM molar concentration of BDT (1,4-benzenedithiol) solution, sonicating for 10 minutes, centrifuging at 6000 rpm for 10 minutes to obtain an Au core with a self-assembled monolayer of BDT, a Raman reporter. Recovered. The recovered Au core on which the self-assembled monolayer was formed was dispersed in 1 mL of deionized water (0.1 nM molar concentration), _{500 μL of 5 mM HAuCl 4} and 2.5 mL of 50 mM HEPES buffer solution of pH 7.2 were added to the dispersion, and stirred at 1000 rpm for 30 minutes. After that, an excess of deionized water was added to terminate the reaction. Subsequently, the Raman active particles prepared by centrifugation at 4000, 3000, and 2000 rpm for 10 minutes were recovered, and stored in 1 ml of 50 mM HEPES buffer.

1, it can be seen that Raman active particles having a shell having surface irregularities are formed by Au fine particles. The average diameter of the prepared Raman active particles was 95 nm, and the average size of the Au particles forming the shell was 22 nm. As shown in FIG. 1, it can be seen that the Au particles protrude and surface irregularities are formed on the shell, and it can be seen that the irregularities due to the projection of the particles are formed omnidirectionally based on the particle center.

2 is a transmission electron microscope photograph of the prepared Raman active particles.

2, it can be seen that the Raman Reporter's self-assembled monolayer is positioned between the Au core and the polycrystalline Au shell made of Au fine particles, and a 0.8 nm-thick nanogap is formed in the entire area of the particles.

3 is a view showing the UV-Vis absorption spectrum of each of the Au core itself (cpre AuNP in FIG. 3), the Au core with a self-assembled monolayer (BDT-treated AuNP in FIG. 3), and prepared Raman active particles. As can be seen from FIG. 3, the Au core and the Au core on which the self-assembled monolayer is formed have substantially similar absorption spectra, but in the case of the prepared Raman active particles, it can be seen that the absorption peak shifts to about 620 nm. In addition, unlike the Au core or the Au core with the self-assembled monolayer formed thereon, it can be seen that the prepared Raman active particles have a very broad absorption peak with a peak half width of 200 nm or more. In addition, it can be seen that the prepared Raman active particles have a very large absorption in the vicinity of about 620 nm, but also have a significant absorption in near-infrared rays of 750 nm or more, specifically 780 nm or more.

4 is a diagram showing the measurement of the Raman spectrum of the prepared Raman active particles themselves, and is a spectrum obtained by irradiating a 532 nm laser (5 mW), a 633 nm laser (5 mW) or a 780 nm laser (5 mW). As can be seen from FIG. 4, when irradiating 633nm light, the highest intensity Raman signal is obtained, but it can be seen that even when irradiating 780nm near-infrared light, a strong Raman signal is still obtained. In addition, as can be seen from FIG. 4, when irradiating light in the visible region, very large basal fluorescence is generated. However, it can be seen that, when irradiated with near-infrared light of 780 nm, significant basal fluorescence does not occur so as to affect the detection signal. Accordingly, when performing a Raman spectroscopy analysis by irradiating near-infrared rays on the Raman active particles according to an embodiment of the present invention, it can be seen that the intensity of the detected signal can be used as the Raman signal intensity without any special signal processing, It can be seen that reliable Raman analysis can be performed. As is known, the signal processing process for removing the basal fluorescence may cause distortion of the Raman signal, which is particularly problematic in quantitative analysis.

5 is a scanning electron microscope photograph (FIG. 5(a)) observed after placing the prepared Raman active particles (α, β, γ) on a silicon substrate and a Raman mapping of the region observed with a scanning electron microscope (780 nm laser, 5mW). As can be seen from Figure 5, it can be seen that the prepared Raman active particles have very uniform Raman activity.

FIG. 6 is a diagram showing an overlapping Raman spectrum of each of Raman active particles α, β, and γ mapped to Raman in FIG. 5. As can be seen from FIG. 6, it can be seen that substantially the same Raman spectrum between Raman active particles is obtained.

Similarly, after obtaining a Raman spectrum of each Raman active particle for 60 prepared Raman active particles, the average value and standard deviation of the peak intensity were measured for one Raman peak, and as a result, the average of 87.6 (au) and 7.5 It was confirmed that it had a standard deviation of (au) and showed extremely uniform Raman activity between particles.

7 is a tau protein as a detection target material, a monoclonal tau antibody having different epitopes as a first receptor and a second receptor, and tau protein A Raman spectrum obtained by using a PBS (Phosphate-Buffered Saline) buffer solution containing a concentration of 1 pg/mL as a sample (Fig. 7(a)) and a Raman map (using a Raman signal of ^{1555 cm -1, Fig. 7 (} b)).

In detail, the Raman active particles on which the first receptor was formed and the substrate on which the second receptor was formed were prepared as follows. Raman active particles in which the first receptor is formed: 1 μM MUA (11-Mercaptoundecanoic acid) was added to 1 mL of the dispersion (particle concentration 1 nM) of Raman active particles prepared as shown in the observation photo of FIG. 1 and left overnight, followed by centrifugation. (3000 rpm, 10 minutes) to recover the particles. Thereafter, 20 μM EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide) and 5 μM NHS (N-hydroxysuccinimide) were added to the recovered Raman active particles and reacted for 15 minutes, followed by centrifugation (3000 rpm, 10 minutes). Raman active particles surface-modified with a carboxyl group were recovered. Thereafter, the Raman active particles surface-modified with a carboxyl group were added to 1 mL of a 50 mM HEPES buffer solution (pH 7.4) containing a monoclonal tau antibody (first receptor) at a concentration of 10 μg/mL, and then reacted for 2 hours, and the same BSA (Bovine serum albumin) was added to the solution so as to be 1% by weight, reacted for 1 hour, and then centrifuged (3000 rpm, 10 minutes) to prepare Raman active particles in which the first receptor and blocking molecule were formed. The prepared Raman active particles (hereinafter, tau probe) were dispersed and stored in 1 mL of 50 mM HEPES buffer.

Substrate on which the second receptor was formed: The substrate having a gold (Au) surface was immersed in 1 mM 11-Mercaptoundecanoic acid (MUA) solution overnight, and then washed with ethanol/deionized water. Then, the recovered substrate was immersed in a solution of 200mM EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide) and 50mM NHS (N-hydroxysuccinimide) in deionized water, reacted for 30 minutes, and then washed again with deionized water. , To prepare a gold substrate surface-modified with a carboxyl group. 20 mL of a 50 mM HEPES buffer solution (pH 7.4) containing a monoclonal tau antibody (second receptor) having an antigenic determinant different from the first receptor at a concentration of 50 μg/mL was applied to the gold substrate surface-modified with the prepared carboxyl group, and After reacting for 1 hour, the substrate was washed with 1% by weight of BSA (Bovine serum albumin) solution to prepare a substrate (hereinafter, referred to as tau substrate) on which the second receptor was formed.

Sample detection was performed as follows. A 0.1 mL sample was dropped on the prepared tau substrate (substrate size = 1cmx1cm, active surface area = 1μmx1μm) and reacted for 60 minutes, and then the unreacted sample was washed and removed using deionized water. Thereafter, 200 μL of a 50 mM HEPES buffer solution containing a tau probe at a concentration of 0.5 nM was dropped on the tau substrate reacted with the sample, and then reacted for 1 hour, and then the unreacted tau probe was washed and removed using deionized water.

Raman analysis was performed as follows. 780nm laser excitation light, 5mW laser power, NA0.75 lens, 1 second acquisition time, 1μm mapping pitch, 40μmx40μm mapping area size, 1555 cm ^-1 Raman signal mapping image

8 is a schematic diagram showing a structure in which a tau protein is bound to a second receptor of a tau substrate, and a tau probe is bound to the tau protein bound to the substrate. As shown in the example illustrated in FIG. 8, the detection target material is fixed to the active surface by the second receptor, and the Raman active particles may be fixed to the detection target material fixed to the active surface by the first receptor. Each of the points at which the Raman signal is detected in the Raman map of FIG. 7 (square dots of FIG. 7) corresponds to a region in which the bonding structure of the active surface-detection target material-Raman active particle as shown in FIG. 8 is formed.

FIG. 9 shows the same tau substrate and tau probe as in FIG. 7 and performs the same Raman analysis, but the concentration of tau protein is 1 pg/mL, 100 fg/mL, 10 fg/mL, or 1 fg/mL. It is a diagram showing the Raman mapping results performed by samples of a PBS (Phosphate-Buffered Saline) buffer solution containing as a sample. As can be seen from FIG. 9, as the concentration of the analyte in the sample increases, the number of analytes fixed between the active surface and the Raman active particles increases by combining the active surface and the Raman active particles, respectively, and the Raman map as shown in FIG. The number of points at which the Raman signal is detected increases.

10 is a log value of the molar concentration of tau protein obtained by analyzing a sample (standard sample) with different tau protein concentrations as shown in FIGS. 7 and 9-a reference graph of the summation intensity (Tau in PBS = of the standard sample) A measurement value, a straight line = a reference graph) and a result of detecting cerebrospinal fluid (Tau in aCSF in FIG. 10) or plasma (Tau in plasma in FIG. 10) collected from the human body as a sample. At this time, the summation intensity is the intensity obtained by summing the maximum intensity at each of the points at which the Raman signal is detected in the Raman map measured using each sample, and when tau protein is detected in the standard sample and the sample collected from the human body, the Raman mapping area However, the Raman analysis conditions, the sample detection conditions (the concentration and amount of the applied Raman active particle dispersion, etc.) were all the same.

As can be seen in Figure 10, in the log value of the molar concentration of tau protein-the semi-log graph of the sum strength, the log value of the molar concentration of tau protein (MC) and the sum strength (I _sum ) are MC = aI _sum + b ( It can be seen that a and b have a linear relationship of real number), and the coefficient of determination (R ² ) is 0.983, and it can be seen that extremely good linearity is maintained in a very wide concentration range of ^{10 -2} to 10 ^{6 fM, and detection} With a limit (LOD) of about 20aM, it can be seen that even one tau protein has a detectable sensitivity.

In addition, in FIG. 10, a result of quantitative detection of tau protein concentration in a cerebrospinal fluid sample (Tau in aCSF in FIG. 10) or a plasma sample (Tau in plasma in FIG. 10) using a reference graph, and tau protein actually contained in each sample. As a result of comparing the concentration, it was confirmed that tau protein was quantitatively detected with an accuracy of about 97%.

As described above, the present invention has been described by the specific matters and limited embodiments and drawings, but this is provided only to help a more general understanding of the present invention, and the present invention is not limited to the above embodiments, and the present invention is Those of ordinary skill in the relevant field can make various modifications and variations from these descriptions.

Therefore, the spirit of the present invention is limited to the described embodiments and should not be defined, and all things that are equivalent or equivalent to the claims as well as the claims to be described later fall within the scope of the spirit of the present invention. .

Claims (14)

A spherical plasmonic metal core, a plasmonic metal shell having surface irregularities, a self-assembled monolayer formed between the core and the shell and located between the core and the shell and including a Raman reporter, and located on the surface of the plasmonic metal shell, It includes a first receptor capable of specifically binding to a substance to be detected, wherein the plasmonic metal core and the plasmonic metal shell are metals that generate surface plasmon by interaction with light, respectively, Raman active particles; A detection method for detecting a detection target substance in the sample by contacting it with a sample that may contain a target substance and irradiating excitation light. The method of claim 1,
The excitation light is a near-infrared ray detection method. The method of claim 1,
a) contacting the sample with the substrate on the surface of the second receptor that specifically binds to the substance to be detected;
b) contacting the Raman active particles with the substrate in contact with the sample;
c) obtaining a Raman spectrum by irradiating excitation light on a substrate in contact with the Raman active particles; And
d) detecting the presence or absence of the detection target substance and the concentration of the detection target substance in the sample based on the Raman spectrum;
Detection method comprising a. The method of claim 2,
When irradiating the excitation light, the step of obtaining a Raman map of the Il Raman signal by performing Raman mapping with a preset area; Including, a sample based on the sum intensity obtained by summing the maximum intensity of the Il Raman signal on the Raman map A detection method for detecting the presence or absence of a detection target substance and the concentration of the detection target substance. The method of claim 4,
The detection method that the concentration of the substance to be detected is calculated by Equation 1 below.
(Equation 1)
MC= aI _sum + b
(In Equation 1, MC is the logarithm of the molar concentration of the substance to be detected, and I _sum is the summation strength) The method of claim 5,
The detection method in which the limit of detection (LOD) of Equation 1 is 20 aM or less. The method of claim 3,
The detection method further comprising the steps of removing the unreacted sample after step a) and removing the unreacted Raman active particles after step b). The method of claim 1,
The detection target material is one or two or more selected from lesion-labeled biomaterials, pathogens, proteins, nucleic acids, sugars, drugs, and biochemicals having lesion specificity. The method of claim 4,
The Raman map is a detection method in which basal fluorescence is not removed. The method of claim 1,
The plasmonic metal shell includes plasmonic metal fine particles having an average size of 0.1D to 0.6D based on the diameter (D) of the metal core, and a detection method having surface irregularities due to the plasmonic metal fine particles. The method of claim 10,
In the plasmonic metal shell, the inner surface shape of the shell in contact with the self-assembled monolayer is spherical. The method of claim 1,
The self-assembled monolayer has a thickness of 0.5 to 2.0 nm. As a method of detecting a lesion marker for disease diagnosis,
Contacting a substrate on which a second receptor specifically binds to a lesion labeling material is located on a surface and a sample derived from a living body that may contain the lesion labeling material;
A substrate in contact with a biological sample and a spherical plasmonic metal core, a plasmonic metal shell having surface irregularities, a self-assembled monolayer and the plasma that are bonded to each of the core and shell and are located between the core and the shell and include a Raman reporter. The plasmonic metal core and the plasmonic metal shell are metals that generate surface plasmon by interaction with light and contain a first receptor that is located on the surface of the monadic metal shell and can specifically bind to a substance to be detected. Washing after contacting the Raman active particles;
Irradiating near-infrared excitation light on a substrate in contact with the Raman active particles and performing Raman mapping with a preset area to obtain a Raman map of an E Raman signal; And
Detecting the presence or absence of a lesion labeling substance in a biological sample and a concentration of the lesion labeling substance on the basis of a summation intensity obtained by summing the maximum intensity of the Raman signal on the Raman map;
Detection method comprising a. A spherical plasmonic metal core, a plasmonic metal shell having surface irregularities, a self-assembled monolayer bonded to each of the core and shell and positioned between the core and the shell and including a Raman reporter, and located on the surface of the plasmonic metal shell And a first receptor capable of specifically binding to a substance to be detected, and the plasmonic metal core and the plasmonic metal shell each contain Raman active particles, which are metals that generate surface plasmon by interaction with light. A surface-enhanced Raman scattering-active reagent; And
A substrate having a second functional group that specifically binds to the substance to be detected is located on the surface;
A detection device comprising a.

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